1. Field of the Invention
The present invention relates to a particle analyzing apparatus capable of detecting a focusing state with respect to a flow section of a measuring optical system.
2. Related Background Art
In a conventional particle analyzing apparatus employed, for example, in a flow cytometer, a particle to be examined, such as a blood cell, suspended in a sheath liquid and flowing through a flow section of a minute rectangular cross section of for example 200 .mu.m .times.200 .mu.m at the center of a flow cell, is irradiated with light such as a laser beam, and information on the particle, such as shape, dimension, refractive index, etc., can be obtained by measuring light forward or laterally scattered thereby. Also in a specimen which can be dyed with a fluorescent material, important information for particle analysis can be obtained by measuring the fluorescent light in the laterally scattered light, which is scattered in a direction substantially perpendicular to the irradiating light.
For achieving a correct measurement in a flow cytometer or the like, the light beam has to be correctly focused on the particle to be examined or on the vicinity thereof by means of a measuring objective lens, in order to avoid pseudo signals coming from objects other than the particle to be examined, and, for this purpose, the measuring objective lens requires exact focusing. In a conventional apparatus, however, focusing is conducted manually with a standard sample under visual observation prior to the actual measurement. However, such a focusing operation is not only cumbersome, but is also inaccurate due to differences between examiners.
Also an eventual aberration in focusing, appearing in the course of measurement, cannot be detected. Consequently, since the presence of pseudo signals in the course of measurement cannot be detected, a certain ambiguity about the reliability of the obtained data remains.
In addition, the focusing operation has to be repeated whenever a nozzle or the flow cell is replaced and an additional time is required for this focusing operation. On the other hand, a very weak fluorescent signal has to be intensified to allow fluorescent measurement, and, for this purpose, it has been proposed to employ a photomultiplier as the detector for the fluorescent light, in order to improve the light-emitting efficiency of the fluorescent material. In addition to the above, increasing the power of the irradiating light source and improving the light condensing power of the measuring objective lens have also been proposed. The condensing power of the objective lens can be improved by the use of a larger aperture, but this leads to a smaller focal depth. With such a smaller focal depth, even a slight change in the distance between the flow section and the objective lens gives rise to introduction of noise signals from other objects, thereby prohibiting exact measurement. In this manner, the conventional apparatus not only requires a cumbersome focusing operation, but also cannot provide a sufficient precision of analysis due to an insufficient intensity of the fluorescent signal.
In order to eliminate the above drawbacks, the present applicant filed copending U.S. Ser. No. 818,263 In the apparatus of this copending application, a focusing light beam irradiates front and rear walls of a flow section of a flow cell along an optical path oblique to the optical axis of a measuring objective lens. The beams reflected by the front and rear walls are received by devided elements of a photosensor. A focusing state is obtained by simultaneously moving both the measuring objective lens and a focusing optical system until output signals, from the divided elements are the same.
In this apparatus, a sufficiently precise focusing state is normally obtained. However, since the focusing state is measured by a comparison between the output signals from the divided elements, when the interior of the flow section of the flow cell is soiled to a considerable degree and the intensity of the reflected light is thus decreased, it is difficult to compare the output signals. In addition, when a difference occurs in the reflectances between the front and rear walls of the flow sections due to the soiled surfaces thereof, correct detection cannot be performed.